Injectable peripheral nerve specific hydrogel

ABSTRACT

The present invention relates to a peripheral nerve-specific hydrogel material, which is deliverable in a minimally invasive fashion, sustains the growth of neurons, and speeds recovery following surgical reconstruction.

1. PRIORITY CLAIM

This application is a continuation of International Patent ApplicationNo. PCT/US2014/021065, filed Mar. 6, 2014 and claims priority to U.S.Provisional Application Ser. No. 61/773,615, filed Mar. 6, 2013, to bothof which priority is claimed and the contents of both of which areincorporated herein in their entireties.

2. INTRODUCTION

The present invention relates to a peripheral nerve-specific hydrogelmaterial, which is deliverable in a minimally invasive fashion, sustainsthe growth of neurons, and speeds recovery following surgicalreconstruction.

3. BACKGROUND OF THE INVENTION

Neuromuscular denervation is a common consequence following peripheralnerve injury. Functional outcomes following repair are oftendisappointing as the capacity of motor axons to regenerate is decreasedwith prolonged denervation. Despite advances in microsurgical techniqueand extensive studies on nerve repair, presently used reinnervationmethods produce moderate results and full functional recovery afterperipheral nerve injury is seldom achieved.

In cases where anastomosis of the nerve is not possible (“criticallysized defect”) the current clinical “gold standard” is often nerveautografting. However, autograft harvest is associated with morbidity atthe donor site including pain, sensitivity, or loss of sensation andapproximately 50% of patients do not regain function following nerveautografting. Allogeneic graft materials have also been suggested.However, fresh allogeneic tissue is subject to an undesirable immuneresponse from the host in the absence of immunosuppression.

For these reasons, a number of alternative approaches have beensuggested. These have included both synthetic and biologically derivedguidance conduits and hydrogel delivery systems. A wide range ofsynthetic polymers have been examined for construction of nerve guidanceconduits, both with and without cell scale features which either mimicthe natural extracellular matrix or provide guidance cues for axonalelongation. However, synthetic nerve guidance conduits are desirablyspecifically tailored not only to support cellular growth, but also toallow for nutrient diffusion, and to degrade with new tissue formationwithin the conduit. Long-term implantation of slow degrading syntheticbiomaterials is also often associated with a detrimental foreign bodytype reaction which can hinder recovery.

Various biologically derived materials have been investigated for thefabrication of nerve guidance conduits. The use of a number ofindividual extracellular matrix (ECM) proteins including collagen,fibronectin, and laminin as well as other biologic materials have beensuggested for the fabrication of nerve guidance conduits into simpletubes, or tubes with intraluminal structures intended for guidance oftissue ingrowth. Many of these approaches have been shown to improveoutcomes in animal models, although only over relatively short lengths.Others have suggested the use of decellularized allograft nerve tissuesas scaffolds for reconstruction of peripheral nerves due to maintenanceof the native tissue architecture and functional molecules in theirrelative tissue specific constituent proportions. However, not all ofthe proposed decelluarlization methods have been shown to be effectivefor removal of sufficient cellular content and maintenance of tissuestructure, resulting ineffective recovery in some studies.

4. SUMMARY OF THE INVENTION

The present invention relates to a decellularized peripheral nervespecific scaffold which can be formulated into an injectable hydrogelform. It is based, at least in part, on the discovery of adecellularized tissue which substantially lacks immunogenic cellularcomponents but retains sufficient amounts of nerve specific componentsto be effective at supporting nerve regrowth and reducing or preventingmuscular atrophy. In certain non-limiting embodiments, thedecellularized tissue scaffold is formulated into a hydrogel through theuse of enzymatic degradation. These hydrogels have been shown to benon-cytotoxic to neurons and also support neuronal outgrowth of culturedcells. When injected into injured recurrent laryngeal nerves in a caninemodel, the hydrogel was shown to improve reinnervation and slow atrophyof the laryngeal muscles as compared to surgical correction withoutinjection.

5. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-F. Confirmation of decellularization. Decellularization wasconfirmed by the absence of nuclei in DAPI stained sections ofdecellularized nerve (B). Native nerve shown in (A). Luxol fast bluestaining confirmed the removal of potentially immunogenic cellularmyelin (D). Native nerve shown in (C). DNA content was assessed byPicoGreen Assay and shown to be reduced by nearly 85% to levels (average175 ng DNA/mg scaffold dry weight) below those reported for mostcommercially available ECM scaffold materials (E). DNA was further shownto be reduced on agarose gel analysis (F).

FIG. 2A-D. Electron microscopy (A,C) and histologic evaluation (B,E) ofstructure. Under SEM, the decellularized sciatic nerves (B) werecharacterized by an ultrastructure similar to that of native tissue (A).In cross section, distinct nerve bundles can be observed, as canindividual structures including the epineureal connective tissues anddense, intact perineurium surrounding each individual nerve bundle. Theepineurium is less dense and larger pores are observed than in thenative tissue. A number of individual reticular fibers can also beobserved. These larger and smaller fibers likely represent the collagenI and III which comprise the majority of the extracellular matrix of thenative epineurium. The endoneureal structure is slightly disrupted ascompared to native tissue. However, the areas previously occupied byindividual nerve fibers (basal lamina) can be observed, and remainhighly parallel within the tissue. This is also evident in longitudinalsection, where aligned channels and connective tissues can be observedmore clearly. Intact blood vessels were also observed in thedecellularized samples. These findings were supported by histologicstaining. Hematoxylin and eosin stained samples (B,D) in cross sectionwere characterized by a diffuse epineurium, a dense and intactperineurium and basal lamina devoid of cells. Longitudinal sectionsfurther confirmed the SEM findings, and were characterized by alignedconnective tissue within the epineurium, and patent spaces indicative ofbasal lamina devoid of axonal and supporting cells.

FIG. 3. Maintenance of specific ECM components. Immunolabeling confirmedthe presence of collagen I, III, and IV within the decellularizedsamples. These components were observed present in an architecture thatresembled that of native nerve. However, the intensity of the stainingwas less and the architecture slightly disrupted as compared to nativetissue.

FIG. 4A-D. Hydrogel formation. Enzymatically digested samples of thedecellularized nerve described above were neutralized and reconstitutedat multiple concentrations and placed into an incubator at physiologictemperature to induce gelation (A,B). Hydrogel formation was improved atconcentrations of 12.5+mg/mL. SEM of hydrogels demonstrates a highlyfibrous architecture of hydrogel (C,D).

FIG. 5A-C. Nerve ECM digest is non-cytotoxic and promotes neuriteoutgrowth. When a neuronal cell line was exposed to the digested ECM,neurite extension was seen as early as Day 1 of culture and continuedwith increasing extension length for up to 3 days in culture. Longertime periods were not examined. Day 0 (A), Day 1 (B), and Day 3 (C) areshown. Neurite extension following exposure to ECM digest was equivalentto results obtained for neurons exposed to neurite outgrowth media(positive control) suggesting the decellularized nerve hydrogel promotesnerve outgrowth.

FIG. 6. Gross morphology of control (left panel) and treated (rightpanel) sciatic nerves at 21 days post-surgery. There was little to notissue growth within empty conduit compared to conduit filled with nervespecific (NS)-ECM. Top panels show conduit before explant and bottompanels show tissue formed within the conduit (conduit removed).

FIG. 7A-G. Qualitative assessment of decellularization. Few cellsremained while maintaining much of the structure. The Tubular structureremained after decellularization. (A & B) H&E, Native and Decell, (C &D) Luxol Fast Blue for Myelin, Native and Decell, and (E & F) Dapistain, Native and Decell, (G) Picogreen assay for DNA content showsdsDNA content of the decellularized tissue (158.07±34.53 ng/mg) wassignificantly decreased as compared to native tissues (1043.65±291.20ng/mg).

FIG. 8A-N. Immunohistology, Electromicroscopic and Biochemical analysisfor major extracellular matrix molecules. Native and decellularizednerve tissue stained for (A) Collagen I native, (B) Collagen I NS-ECM,(C) Collagen III native, (D) Collagen III NS-ECM, (E) Collagen IVnative, (F) Collagen IV NS-ECM. (G and H) Electromicroscopic appearanceof intact nerve. (I and J) Electromicroscopic appearance ofdecellularized nerve. (K-N) Biochemical assays for hydroxyproline, GAG,NGF, and CNTF content.

FIG. 9A-D. (A) Degradation products of decellularized nerves wereintroduced at different concentrations to primary neurons over fourdays. (B) Images from neurite outgrowth with pepsin control and positive(b27 supplement) control and nerve and urinary bladder matrix (UBM) at500 ug/ml. (C and D) Digested decellularized nerve products were formedinto a stable gel. Concentration of gel was 15 mg/mL.

FIG. 10A-D. (A) Intraoperative photo of silicon conduit placement afteranastomosis of transected recurrent laryngeal nerve (RLn). (B) Compoundmuscle action potential amplitude measured at the vocalis muscle aftersuper-stimulation of the RLn. (C) Duration between stimulation of theRLn and recorded action potential at the vocalis muscle. (D) Musclefiber diameter of the posterior cricoarytenoid muscle. All threemeasurements were taken after 6 months of recovery, post-surgery.

FIG. 11. NS-ECM injection. NS-ECM was injected within a silicone conduitto support the grafting of the first cervical nerve to the Rin threemonths post-injury.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a peripheral nerve-specific hydrogelmaterial, which is deliverable in a minimally invasive fashion, that iscapable of sustaining the growth of neurons and promoting recoveryfollowing surgical reconstruction.

In certain non-limiting embodiments, the present invention provides fora nerve tissue-specific decellularized scaffold and hydrogel forperipheral nerve repair, wherein the scaffold material is derivedthrough the decellularization of a peripheral nerve. In non-limitingembodiments, as exemplified below, a portion of the peripheral nerve maybe used to form the scaffold. Said peripheral nerve can be obtained froman autologous or a non-autologous source. In one non-limitingembodiment, said peripheral nerve is obtained from a non-autologoussource, such as syngenic, allogeneic or xenogeneic source which may beof the same or a different species, such as a human or a non-humananimal such as a non-human primate, a dog, a cat, a horse, a cow, asheep, a goat, or a pig. In non-limiting embodiments, the peripheralnerve is a sciatic nerve, femoral nerve, ulnar nerve, median nerve,musculocutaneous nerve, common peroneal nerve, sural nerve or othermotor or sensory nerve. In one specific non-limiting embodiment, theperipheral nerve is an equine sciatic nerve.

The scaffold material retains sufficient nerve specific components so asto effectively support nerve repair. The hydrogel form of the scaffoldis injectable at sites of nerve repair, and has been shown effective inpromoting nerve functional recovery and in reducing muscular atrophywhen used at sites of nerve repair in the recurrent laryngeal nerve.

In certain non-limiting embodiments, the present invention provides oneor more of the following advantages:

a method of decellularization which reduces immunogenic cellularcomponents to a minimal level while retaining a large number ofperipheral nerve-specific components;

a hydrogel which is peripheral nerve specific (the inventors are notaware of any other peripheral nerve specific decellularized hydrogel,and data supports the concept that tissue specific scaffolds generate asuperior response to generic xenogeneic scaffolds); and

the material has been demonstrated to promote recovery followinginjection around surgically reconstructed peripheral nerves in aclinically relevant large animal model.

Accordingly, in certain embodiments the invention provides for ahydrogel comprising a decellularized peripheral nerve scaffold that hasbeen enzymatically degraded to form a hydrogel, wherein the hydrogelpromotes peripheral nerve repair. In specific non-limiting embodiments,the hydrogel is injected into or comprised in a conduit, as is known inthe art to facilitate or guide the regrowth of nerves. The conduits canbe absorbable or non-absorbable. Suitable conduits include, but are notlimited to, three types of bioabsorbable conduit that have been approvedby the US Food and Drug Administration: conduits constructed ofcollagen, polyglycolic acid, or caprolactone (Deal 2012).

In a non-limiting embodiment, peripheral nerve extracellular matrix(ECM) may be prepared as follows. A peripheral nerve, for example asciatic nerve, may be harvested and then frozen for at least 16 h at−80° C. The epineurium may be stripped and the tissue quarteredlongitudinally and cut into lengths of <5 cm. Decellularization may thenbe performed as previously described by Medberry (2013). For example,the decellularization process may comprise a series of agitated washes:water (type 1), 0.02% trypsin/0.05% EDTA (60 min at 37° C.), 3.0% TritonX-100 (60 min), water rinse (type 1, repeated until agitation no longerproduced bubbles), 1M sucrose (15 min), 4.0% sodium deoxycholate (60min), 0.1% peracetic acid/4% ethanol (120 min), 1×PBS (15 min), water(15 min), water (15 min), 1×PBS (15 min). Following treatment samplesmay be frozen (−80° C.) and then lyophilized.

In non-limiting embodiments, enzymatic degradation product and ECMhydrogel may be prepared as follows. Lyophilized scaffold materials maybe powdered using a Wiley mill through a 40 mesh screen. The powderedmaterial may be solubilized at a concentration of 20 mg/mL in a solutioncontaining 2.0 mg/mL pepsin in 0.01 N HCl at a constant stir rate for 48h. The ECM digest solution may then be frozen at −20° C. until use.Enzymatic digestion may be stopped by raising the pH of the solution to7.4 using NaOH and diluting the solution to the desired concentrationwith PBS. Gellation of the neutralized digest may be induced byincreasing the temperature of the gel into the physiologic range 37° C.

Alternative decellularization processes and methods of generating ECMdegradation product are known in the art and may be used where theresulting product is at least 50% or at least 80% or at least 90% asperipheral nerve specific as the hydrogel prepared according to themethod set forth above. Other potential variations upon the hydrogel mayinclude addition of bioactive components or therapeutic agents.Therapeutic agents within the hydrogel can be used in various ways. Thetherapeutic agent can be released from the hydrogel. For example, ananti-inflammatory drug can be released from the hydrogel to decrease animmune response. Additionally or alternatively, the therapeutic agentcan be substantially remain within the hydrogel. For example, achemoattractant can be maintained within the hydrogel to promotecellular migration and/or cellular infiltration into the hydrogel. Atleast one therapeutic agent can be added to the hydrogel before it isinjected into a conduit. Suitable therapeutic agents can include anysubstance that can be coated on, embedded into, absorbed into, adsorbedonto, or otherwise attached to or incorporated onto or into the hydrogelthat would provide a therapeutic benefit to an intended recipient.Suitable therapeutic agents include, but are not limited to,antimicrobial agents, growth factors, emollients, retinoids, and topicalsteroids. Each therapeutic agent may be used alone or in combinationwith others. In one non-limiting embodiment, the hydrogel comprisesneurotrophic agents or cells that express neurotrophic agents, whichhydrogel is used for nerve repair.

The therapeutic agent can be a growth factor, such as a neurotrophic orangiogenic factor, which optionally may be prepared using recombinanttechniques. Suitable growth factors include, but are not limited to,basic fibroblast growth factor (bFGF), acidic fibroblast growth factor(aFGF), vascular endothelial growth factor (VEGF), hepatocyte growthfactor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2),platelet derived growth factor (PDGF), stromal derived factor 1 alpha(SDF-1 alpha), nerve growth factor (NGF), ciliary neurotrophic factor(CNTF), neurotrophin-3, neurotrophin-4, neurotrophin-5, pleiotrophinprotein (neurite growth-promoting factor 1), midkine protein (neuritegrowth-promoting factor 2), brain-derived neurotrophic factor (BDNF),tumor angiogenesis factor (TAF), corticotrophin releasing factor (CRF),transforming growth factors α and β (TGF-α and TGF-β), interleukin-8(IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF),interleukins, and interferons. Commercial preparations of various growthfactors, including neurotrophic and angiogenic factors, are availablefrom R & D Systems, Minneapolis, Minn.; Biovision, Inc, Mountain View,Calif.; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel; and CellSciences®, Canton, Mass.

Additionally or alternatively, the therapeutic agent can be anantimicrobial agent. Suitable antimicrobial agents include, but are notlimited to, isoniazid, ethambutol, pyrazinamide, streptomycin,clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin,rifampin, azithromycin, clarithromycin, dapsone, tetracycline,erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B,ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin,lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir,trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir,iatroeonazole, miconazole, Zn-pyrithione, and silver salts such aschloride, bromide, iodide and periodate.

The therapeutic agent can be an anti-inflammatory agent. Suitableanti-inflammatory agents include, but are not limited to, a NSAID, suchas salicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen, sodium salicylamide; an anti-inflammatorycytokine; an anti-inflammatory protein; a steroidal anti-inflammatoryagent; and an anti-clotting agent, such as heparin. Other drugs that maypromote wound healing and/or tissue regeneration may also be included.

Another variation may include polymeric components or additionalbiologic components in addition to the hydrogel. Another variation wouldinclude the hydrogel which has been seeded with cells prior to or at thetime of injection. The cells that are integrated may remain after thehydrogel has fully disintegrated within the conduit. However, themicrointegrated cells may also be merely cells that act as precursors tothe final tissue that is formed when the hydrogel has fully degraded.Cells may be autologous (obtained from the intended recipient), from anallogeneic or xenogeneic source or from any useful cell line, including,but not limited to, stem cells or precursor cells (cells that candifferentiate into another cell type) that are capable of cellulargrowth, remodeling, and/or differentiation. Suitable cells that can beincorporated onto or into the hydrogel include, but are not limited to,stem cells, precursor cells, smooth muscle cells, skeletal myoblasts,myocardial cells, endothelial cells, fibroblasts, chondrocytes andgenetically modified cells. Various commercially available cell linesinclude Clonetics® Primary Cell Systems (Lonza Group, Inc.,Switzerland), ATCC.

Non-limiting examples of uses of the hydrogel of the invention includethe following.

In certain non-limiting embodiments, the hydrogel may be injected withina conduit to be used to bridge critically sized defects. A “criticallysized defect”, as used herein, is a defect not amenable to joininganastomosis, for example, but not limited to, a gap of at least about 10mm, or a gap of at least about 15 mm, or a gap of at least about 20 mm.

In certain non-limiting embodiments, the hydrogel may be used to supportdirect peripheral nerve repair either soon after injury (acute) or aftera period of delay (chronic), e.g., at least about two weeks delay, atleast about 1-month delay, at least about 2-months delay, at least about3-months delay, at least about 4-months delay, at least about 5-monthsdelay, at least about 6-months delay, at least about 12-months delay, orat least about 24-months delay, and/or up to about 2-years delay, up toabout 3-years delay, up to about 4-years delay, or up to about 5-yearsdelay. Delays often occur prior to nerve repair to allow soft tissue andorthopedic injuries to heal.

In certain non-limiting embodiments, the hydrogel may be used to supporta peripheral nerve graft used to reinnervate a denervated nerve eithersoon after injury (acute) or more commonly after a period of delay(chronic), e.g., at least about two weeks delay, at least about 1-monthdelay, at least about 2-months delay, at least about 3-months delay, atleast about 4-months delay, at least about 5-months delay, at leastabout 6-months delay, at least about 12-months delay, or at least about24-months delay, and/or up to about 2-years delay, up to about 3-yearsdelay, up to about 4-years delay, or up to about 5-years delay. In onenon-limiting embodiment, the delay is about 3 months.

In certain non-limiting embodiments, the hydrogel may be used to bridgecritical gaps in peripheral nerves. These gaps could arise as a resultof traumatic injury or iatrogenic injury during surgery for exampletumour removal.

7. EXAMPLES Example 1

Equine sciatic nerve was harvested at the Cornell University College ofVeterinary Medicine following euthanasia of adult animals for reasonsunrelated to the sciatic nerve. The tissue was then frozen for at least16 h at −80° C. The epineurium was stripped and the tissue was quarteredlongitudinally and cut into lengths of <5 cm. Decellularization was thenperformed as previously described by Medberry (2013). Briefly, thedecellularization process consisted of a series of agitated washes:water (type 1), 0.02% trypsin/0.05% EDTA (60 min at 37° C.), 3.0% TritonX-100 (60 min), water rinse (type 1, repeated until agitation no longerproduced bubbles), 1M sucrose (15 min), 4.0% sodium deoxycholate (60min), 0.1% peracetic acid/4% ethanol (120 min), 1×PBS (15 min), water(15 min), water (15 min), 1×PBS (15 min). Following treatment sampleswere frozen (−80° C.) and then lyophilized.

Enzymatic degradation products were generated as previously described.Briefly, lyophilized scaffold materials were powdered using a Wiley millthrough a 40 mesh screen. The powdered material was solubilized at aconcentration of 20 mg/mL in a solution containing 2.0 mg/mL pepsin in0.01 N HCl at a constant stir rate for 48 h. The ECM digest solution wasthen frozen at −20° C. until use in subsequent experiments. Enzymaticdigestion was stopped by raising the pH of the solution to 7.4 usingNaOH and diluting the solution to the desired concentration with PBS.Gellation of the neutralized digest is induced by increasing thetemperature of the gel into the physiologic range 37° C.

Example 2

Animals (Sprague Dawley rats, female 240-280 g) were anesthetized and anapproach made through the lateral thigh to the sciatic nerve. The nervewas transected proximal to the bifurcation, The nerve ends were drawn 1mm into a 17 mm silicone conduit and secured with 9/0 ETHILON™ NylonSuture to create a critical gap defect of 15 mm. The conduit was filledwith either nerve specific (NS)-ECM hydrogel or left empty. The ECM wasprepared from a canine source. Two minutes were allowed for hydrogelformation and the muscle and skin layers were closed.

Regenerating nerve gaps were excised at 21 days, inspected grossly andfixed for immunohistochemistry. Gaps filled with NS-ECM showed improvedaxonal regrowth from the proximal nerve stump compared to empty conduitsboth grossly (FIG. 6) and on immunohistochemical section.

These data support the ability of NS-ECM to bridge critical gaps inperipheral nerves.

Example 3—Nerve-Specific Extracellular Matrix Hydrogel Promotes RecoveryFollowing Reconstruction of the Recurrent Laryngeal Nerve 1. Summary

Damage to the nerves that innervate the larynx, in particular therecurrent laryngeal nerve (RLn) can result in severe consequences forpatients. Permanent RLn impairment may significantly impact quality oflife by increasing vocal effort and reducing voice quality, and in somepatients the condition can become life threatening. A number of clinicalsolutions exist, however functional recovery following these proceduresis slow and often incomplete. Therefore, methods that accelerate orimprove re-innervation following reconstruction of the RLn are ofsignificant clinical interest. This example describes the production,characterization and use of an injectable, peripheral nerve-specificextracellular matrix (NS-ECM) based hydrogel to improve outcomesfollowing reconstruction of the RLn. The hydrogel was tested in a caninemodel of RLn transection and anastimos. The anastimos was enclosed by asilicon conduit and either left empty or injected with either a NS-ECMhydrogel or a non-nerve-specific extracellular matrix, urinary bladdermatrix (UBM) hydrogel. After 6 months, both NS-ECM and UBM significantlyincreased the amplitude of stimulation reaching the vocalis muscle(0.744 mV and 0.719 mV respectively) compared to the conduit alone(0.343 mV). Afterwards, the posterior cricoarytenoid muscle washarvested and muscle fiber diameter was measured. Differences betweenthe control and treatment groups (NS-ECM and UBM) are small butsignificant, e.g., 53.5 μm compared to 58.4 μm and 61.7 μm,respectively. These results show that the NS-ECM hydrogel can providesupportive scaffolding to promote in vivo axonal repair of the RLn.

2. Materials and Methods

2.1. Preparation of Peripheral Nerve ECM

Equine sciatic nerve was harvested following euthanasia of adult animalsfor reasons unrelated to nerve injury or neurological disease. Thetissue was then frozen for at least 16 h at −80° C. The epineurium wasstripped and the tissue was sectioned longitudinally and cut intolengths of <5 cm. Decellularization was performed as described inExample 1.

2.2. Confirmation of Decellularization

Qualitative assessment of DNA content was performed using immunofluorescence staining. Fixation of lyophilized ECM scaffolds wasperformed in 10% formalin. Samples were embedded in paraffin, sectioned,and stained with H&E or with DAPI to verify removal of nuclei.Additional samples were stained using Luxol™ fast blue (registeredtrademark of Rohm and Haas Co.) to determine removal of myelin.Qualitative assessment of DNA content was conducted by digesting the ECMscaffold in 0.1 mg/mL proteinase K solution. The protein was removed byrepeated Trizol extraction and centrifugation (10,000 G) until no whiteprecipitation (protein) was observed at the interface while the aqueouslayer extract was mixed with 3 M sodium acetate and 100% ethanol. Thesolution was frozen using dried ice and centrifuged to produce a DNApellet. The pellet was rinsed with 70% ethanol, centrifuged (10,000 G),and dried. Double-stranded DNA was quantified using PicoGreen followingkit instructions.

2.3 Assessment of Scaffold Architecture

Scaffold architecture was assessed by scanning electron microscopy(SEM), histologic staining, and immunolabeling. For SEM, lyophilizedsamples were fixed in cold 2.5% (v/v) glutaraldehyde in PBS for at least24 h, followed by three washes in PBS. Lipid fixation was performed in1% (w/v) osmium tetroxide (Electron Microscopy Sciences) for 1 hfollowed by three washes in PBS. Fixed samples were then dehydratedusing a graded series of ethanol-water solutions (30%-100%) followed by15 min in hexamethyldisylizane and subsequent air-drying. The driedsamples were mounted onto aluminum stubs and sputter coated with a 3.5nm layer of gold-palladium alloy using a Sputter Coater 108 Auto(Cressington Scientific Instruments). Images were taken with a scanningelectron microscope (JEOL JSM6330f).

For histologic analysis, scaffold materials were fixed in 10% neutralbuffered formalin, embedded in paraffin and sectioned at 5 μm. Sampleswere dewaxed using xylenes and a graded series of ethanol washes(100-70%) then stained using hematoxylin and eosin. Stained samples weredehydrated using the reverse of the procedure above, coverslipped, andviewed under a light microscope (Nikon e600).

Immunolabeling was performed with antibodies specific to ECM componentsindicative of the neuronal basal lamina (collagen IV and laminin) andmore general connective components (collagen I and III). Briefly, afterdeparaffination, all slides were subjected to antigen retrieval byimmersion in 95° C.-100° C. in citric acid solution (10 mM, pH 6.0; 20min) followed by rinsing in a 1× Tris buffered saline/Tween-20 solution(0.1% Tween 20 v/v, pH 7.4; 3 washes, 5 min each). Samples were thenwashed in PBS and treated with a pepsin digestion (0.05% pepsin w/v in10 mM HCl) solution for further antigen retrieval. Samples were blockedagainst nonspecific binding using a solution consisting of 2% horseserum, 1% bovine serum albumin, 0.1% Tween-20, and 0.1% Triton X-100 inPBS for 30 min at room temperature. Primary antibodies were diluted inthe blocking solution and applied to sections overnight at 4° C.Antibodies to collagen I, III, and IV (Sigma) were used at aconcentration of 1/200. Samples were washed in PBS and appropriatefluorescently labeled secondary antibodies (AlexaFluor 488) were appliedfor 30 min at room temperature. All secondary antibodies were diluted1:250 in the blocking solution. Slides were washed in PBS andcoverslipped in the aqueous mounting medium before observation under afluorescent microscope (Nikon e600).

2.4 Generation of Enzymatic Degradation Products

Enzymatic degradation products were generated as described in Example 1.

2.5 Evaluation of Effects of Enzymatic Degradation Products Upon NeuriteOutgrowth

Spinal cord neurons were isolated from embryonic day 14 Sprague-Dawleyrat pups. Spinal cords were collected in cold Hanks' buffered saltsolution without Ca²⁺ and Mg²⁺ (14 170-112, Gibco, Carlsbad, Calif.,USA), minced into pieces approximately 0.5 mm in size and enzymaticallydissociated in 2 ml 0.25% trypsin solution containing 0.05% collagenaseL1 (MP Biomedicals, Solon, Ohio, USA) at 37° C. for 20 min. Celldigestion was inhibited by adding 2 ml SBTI-DNAse solution (0.52 mg/mlsoybean trypsin inhibitor, T-9003; 3.0 mg/ml BSA, A-2153; 0.04 mg/mlbovine pancreas DNAse, D-4263; Sigma). The cell suspension was gentlytriturated and centrifuged at 800×g for 5 min. The resulting pellet wasthen resuspended in plating medium and gently triturated. The platingmedium consisted of 20% horse serum (16 050-130, Gibco), (25 030-081,Gibco), 5 ml HBSS without Ca²⁺ and Mg²⁺ (14 170-112, Gibco) and 9.8 mlDulbecco's modified Eagle's medium with L-glutamine (DMEM; D5648,Sigma). All non-dispersed tissue was allowed to settle before beingdiscarded. Spinal cord neurons were seeded on poly-L-lysine-coatedcoverslips (12.5 μg/ml in H₂O; P1274, Sigma) in plating medium at aplating density of 3×105 cells/mL and allowed to adhere for 4 h inculture conditions of 5% CO₂ and 37° C. After 4 h, the medium wasexchanged with 1 ml serum-free culture medium containing Neurobasal-A(NBA, 10 888-022, Gibco), and 1 mM Glutamax (35 050-061, Gibco). As apositive control, 1×B27 supplement was added to the media. The cellswere maintained in a humidified 5% CO₂ atmosphere at 37° C., with 50% ofthe culture medium changed every 4 days. Three images at 20×magnification were taken per well. The number of cells with neuriteextensions and the length of neurite extensions were counted usingNeuron) (ImageJ, NIH).

2.6 Formation of Hydrogel from Enzymatic Degradation Products

Gelation was induced by adjusting the pH of the pepsin digest to 7.4using 0.1M NaOH and PBS. Neutralization was accomplished by the additionof one-tenth the digest volume of 0.1 N NaOH, one-ninth the digestvolume of 10×PBS, and then diluting to the desired final ECMconcentration. Concentrations between 8 and 15 mg/mL were examined fortheir ability to form a hydrogel. Dilutions were performed on ice andECM pre-gel was cast into a stainless steel ring within a 6 well cellculture cluster. The gel solution was placed in a non-humidified 37° C.and allowed to gel for 1 h. Resultant hydrogels were then investigatedusing SEM as described above.

2.7 Evaluation of Hydrogel Cellular Compatibility

N1E-115 mouse neuroblastoma cells (ATCC No. CRL 2263), were cultured inDMEM (Sigma) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific,Waltham Mass., USA)/1% pen/strep (Sigma) in T-75 flasks. N1E-115 cellsin DMEM with 2.5% FBS/1% pen strep were seeded at a concentration of8,500 cells on the surface of a 6 mg/mL nerve ECM or UBM-ECM gel in a 96well plate. Wells seeded with cells in a media containing pepsin digestinstead of ECM digest were used as controls. Following 18-24 hrs inculture with ECM, 2 μM calcein-AM and 2 μM ethidium homodimer-1 wasadded to each well to evaluate cytotoxicity. Membrane-permeablecalcein-AM, but not ethidium homodimer-1, is hydrolyzed in live cellsthat fluoresce in green and dead cells that bind and activate ethidiumhomodimer-1, but not calcein-AM, fluoresce in red.

2.8 Animal Anesthetic and Surgical Procedures

2.8.1 Ethics Statement

This study was performed in accordance with the PHS Policy on HumaneCare and Use of Laboratory Animals, the NIH guide for Care and Use ofLaboratory Animals, federal and state regulations, and was approved bythe Cornell University Institutional Animal Care and Use Committee(IACUC). Animals were brought into the research unit and given a 7 dayacclimatization period prior to any procedure. Daily record logs ofmedical procedures were maintained. Cages with elevated floors werecleaned daily and disinfected biweekly. The animals were fed twice a dayto maintain proper body condition, and allowed water ad libitum. Grouphousing provided socialization and ample space for exercise.

2.8.2 Animals and Instrumentation

Eleven female Beagle dogs (age 5-7 years, body weight 6.8±0.7 kg, range6.4-8.7 kg) with no history of upper airway disease and normal laryngealfunction, determined endoscopically, were used. Dogs were chosen atrandom and fasted for at least six hours before anesthestheticprocedures. At the conclusion of each procedure, dogs were monitored forone hour before returning to their group housing.

After fasting overnight, each dog was anesthetized with dexmedatomidine(2 mcg/kg IV followed by 2 mcg/kg/hour) and maintained under anesthesiaat a constant expired isoflurane concentration (approximately 1MAC,1.3%). Monitoring consisted of continuous electrocardiogram, pulseoximetry, non-invasive blood pressure, capnography and temperature.

Analgesia was provided with pre-operative Meloxicam (0.2 mg/kg bodyweight) subcutaneously (SQ), followed by meloxicam given as an oralsuspension (0.1 mg/kg body weight), daily for four additional days.

2.8.3 Surgery

Animals were placed in left lateral recumbency and a lateral approach tothe larynx was made in a standard fashion anterior to the rightlinguofacial vein, the subcutaneous fascia was divided and the maintrunk of the right Recurrent Laryngeal nerve (RLn) identified. The RLnwas identified and transected 15 mm inferior to the cricoid. Theseexperiments were performed on the right side of the larynx as there isevidence for reduced functional recovery of left RLn injury compared toright (Woodson (2008)). Endoscopy using a rigid endoscope (2.7 mmdiameter, Olympus) was performed to verify right sided paralysisimmediately after surgery.

2.8.4 Experimental Groups

Animals were randomly (computer generated code) divided into threegroups. In the control group, RLn transection was immediately followedby end-end RLn-RLn anastomosis performed using 3-5 epineural stitches ofnylon 9-0 suture. Care was taken to avoid driving the needle or suturethrough the nerve fascicles. The anastomosis was surrounded by apreplaced empty 7 mm silicone conduit (3.5 mm internal diameter,Silastic; Dow Corning, Midland, Mich.) tagged to the perineurium ateither end. In the two treatment groups the conduit was filled witheither ECM derived from equine motor nerve (NS-ECM); or with ECM derivedfrom Urinary Bladder Matrix (UBM) and introduced into the conduit lumenvia an 18 gauge needle. Hydrogels were brought to room temperature 5minutes prior to injection into the conduit. The incision was thenclosed in layers.

Animals were euthanized at 6 months following implantation and laryngealmuscles and nerves harvested bilaterally for histology andimmunohistochemistry.

2.8.5 Endoscopy

Endoscopy using a rigid endoscope (2.7 mm diameter, Olympus) immediatelyfollowing surgery, and at 2 and 6 months to assess arytenoid movementunder light sedation (dexmedetomidine (1 mcg/kgIV), Jansson (2000);Ducharme (2010)).

2.8.6 Evoked CMAP Detection

Immediately prior to euthanasia, animals were anesthetized andanesthesia was maintained with isoflurane in O₂ via an endrotrachealtube capable of detecting evoked compound motor action potentials (CMAP)from the vocalis muscle following proximal stimulation of the recurrentlaryngeal nerve (NIM EMG Endotracheal tube, (inner diameter 7.0 mm,outer diameter 10.5 mm) Medtronic) (Dralle (2008).

The right RLn was exposed by dissection in the mid cervical region and asingle monopolar needle (Neuroline monopolar, AMBO Inc.) placed adjacentto the RLn 10 cm caudal to the anterior ring of the cricoid cartilage. Asupramaximal pulse (8-14 mA) was applied to the monopolar needle and thecorresponding CMAP recorded at the vocalis muscle (Sierra Wave II,Caldwell Laboratories, Kennewick, Wash.). Presence/absence and peakamplitude of each CMAP was recorded for each of three repetitions. Thisprocedure was repeated on the left side.

Endoscopy was performed during the same anesthetic episode and thedegree of arytenoid abduction at peak inspiration determined under lightsedation (Jansson (2000); Ducharme (2010)).

2.8.7 Immunohistochemistry-Muscle

Posterior cricoarytenoid (PCA) and lateral cricoarytenoid (LCA) muscleswere harvested from explanted larynges and weighed. Collagen Vimmunonohistochemistry was performed on mid sections of left and rightPCA and LCA muscles and minimum fiber (Feret's) diameters determinedusing custom semi-automated software written in Matlab. 8 μm-thickcryosections of acetone-fixed muscle were used for immunohistochemicalanalysis. Cryosections were washed with phosphate buffered salinecontaining 0.05% Tween 20 (PBST) for 3 times (5 min each). Nonspecificstaining was blocked with a mixture of 10% rabbit serum and 2×casein for30 minutes at room temperature. The primary antibody goat anti-type Vcollagen antibody (SouthernBiotech, Birmingham, Ala.) was diluted to1:1,000 in PBS containing 1×casein, and the sections were incubated for1.5 hr at 37° C. Biotinylated rabbit anti-goat IgG (Vector Laboratories,Burlingame, Calif.) was diluted to 1:200 in PBS, and incubated for 30min at room temperature. Finally, streptavidin-Texas Red (MolecularProbe, Life Technologies, Grand Island, N.Y.) was used to visualizepositive staining (used at 1:200 in PBS), and then the sections weremounted in Vectashield containing Dapi (Vector Laboratories). PBST wasused for washing throughout the procedure. Goat IgG was diluted to thesame final concentration as primary antibody was used as a negativecontrol. IHC results were examined and photographed using Olympus AX 70compound microscope.

2.8.8 Histomorphologic Examination-Nerve

Cross-sections were obtained 5 mm proximal and 5 mm distal to theanastomosis, stained with azur 2-methylene blue-safranin and totalnumber of axons, percentage of myelinated axons, number of fascicles andthe number of axons in the largest fascicle delineated usingsemi-automated software (Volocity). Axon number was divided by the areasampled to calculate average myelinated axon density. Samples werestained also with hematoxylin and eosin for histomorphologicexamination.

2.8.9 Data Analysis

Raw CMAP data was exported as a text file and analyzed using customsoftware written in MATLAB to determine peak amplitude and area of theCMAP. Mean values were determined from the three CMAPs recorded on eachside. For each parameter, differences between left and right CMAPs wasdetermined using Wilcoxon-signed rank tests.

3. Results

3.1 Confirmation of Decellularization

Effective decellularization of ECM based biomaterials is essential toremove the majority of immunogenic cellular constituents while leavingthe tissue-specific structural and functional components intact andpromoting an appropriate regenerative response (Brown (2013), Crapo(2011)). A nerve-specific decellularization protocol was used todecellularize equine sciatic nerve (Medberry (2013)). The NS-ECMgenerated was then fully characterized.

Following decellularization, no nuclei were visible in hematoxylin andeosin (H&E) stained sections under light microscopy (FIGS. 7A and 7B).In some samples, a small number of nuclei (2-3 nuclei/40× field) wereobserved when labeled with DAPI (FIGS. 7C and 7D). When present,retained nuclei were observed within the inner most bundles of thetreated tissues. Myelin, a potentially immunogenic axonal component, wasremoved effectively by the decellularization process (FIGS. 7E and 7F,Luxol® fast blue).

dsDNA content was significantly decreased by approximately 85% in thedecellularized tissue (158.1±34.5 ng/mg) was compared to native tissue(1,043.65±291.20 ng/mg (FIG. 7G, Quantitative PicoGreen assay). Thesevalues are consistent with those reported for multiple FDA approved,commercially available ECM scaffold materials (Gilbert (2009)). Resultswere compared to those obtained for an ECM material derived from urinarybladder, prepared as reported in Brown (2006), for comparison.

3.2 Maintenance of Extracellular Matrix Ultrastructure and Components

The maintenance of nerve-specific structural and functional componentswas investigated using electron microscopy, immunofluorescent labeling,and biochemical assays. Decellularized sciatic nerves were characterizedunder scanning electron microscopy (SEM) by an ultrastructure similar tothat of native tissue (FIG. 8A-D). In cross section, distinct nervebundles were observed, with intact epineureum and a dense, intactperineurium surrounding each individual nerve bundle. The epineurium wasless dense and larger pores are were observed than in the native tissue.A number of individual reticular fibers were also observed. These largerand smaller fibers likely represent the collagen I and III whichcomprise the majority of the extracellular matrix of the nativeepineurium. The architecture of the endoneurium was retained but nolonger contained individual nerve fibers (FIG. 8G-J).

Organization of collagen I, III, and IV within the decellularizedtissues was similar to that of native nerve tissue (FIG. 8E-J). Althoughsome structural components were disrupted, the basal lamina was stronglypreserved as evidenced by the distribution of collagen IV (a majorconstituent of the basal lamina) within the decellularized samples.Collagen IV was present both within the endoneurial basal lamina andwithin the perineurium. The intensity of the staining within theendoneurium, however, was less and the architecture slightly disruptedas compared to native tissue.

Biochemical testing was performed to assess the quantity of basic ECMcomponents including hydroxyproline and glycosaminglycans (GAG) as wellas for nerve-specific growth factors including ciliary neurotrophicfactor (CNTF) and nerve growth factor (NGF). The decellularizationprocess increased the hydroxyproline concentration from 34.9±9.1 ug/mgin native tissue to 65.0±9.4 ug/mg in NS-ECM (FIG. 8K). This enrichmentof hydroxyproline content was likely a consequence of removal of othercomponents of the ECM. Conversely, only 60% of initial GAG content waspreserved (native 51.3±8.0 ug/mg to 31.0±16.7 ug/mg) (FIG. 8M).Enzyme-linked immunosorbent assay (ELISA) showed that 26% and 50% of thenative levels of NGF (native 680.9 pg NGF/g tissue±160.8 pg/g; NS-ECM177.0 pg NGF/g ECM±33.2 pg/g) and CNTF, respectively, were conserved inthe nerve ECM scaffold (FIGS. 8L and 8N). Despite a non-nerve tissueorigin, similar levels of NGF (161.0 pg NGF/g UBM±38.5 pg/g) and CNTFwere found in the UBM scaffold ((FIGS. 8L and 8N; and results weresimilar to those previously reported for UBM.

3.3 Bioactivity of Degradation Products

The degradation products of ECM based scaffold materials derived fromdecellularization of multiple tissues possess bioactivity including theability to promote chemotaxis, proliferation and cell differentiation.Once decellularized, NS-ECM was degraded for processing into a hydrogelas described in Medberry (2013). Briefly, NS-ECM was milled into apowder and digested in a solution of 1 mg/mL pepsin in 0.01 M HCl. Thedigest solution was then neutralized using 0.1 M NaOH, 10× and 1×PBS atvarying concentrations.

Bioactivity of the degradation products were analyzed through a neuriteoutgrowth assay. Primary spinal cord neurons were harvested from E14Sprague-Dawley rat embryos. Cells were then plated and mediasupplemented with a range of concentrations of NS-ECM and UBM digests(125, 250, 500 ng/mL). Neurite outgrowth was assessed at time points 1,2, and 4 days post-plating using an ImageJ based algorithm as previouslyreported. Results were compared to media supplemented with a neuronsurvival and growth supplement (B27, positive control), media alone(negative control), or media containing neutralized pepsin at aconcentration equal to that of the digested ECM treatment groups (FIG.9A-D). A dose dependent increase in neurite outgrowth was observed atall time points with increasing neurite outgrowth observed to correspondto increasing ECM supplementation.

3.4 Formation of Injectable Hydrogel

The decellularized tissue has been enzymatically digested and thosenerve-specific degradation products were found to promote neuronaloutgrowth. Those same degradation peptides can be formed into athermally-sensitive hydrogel. The pepsin digest spontaneouslypolymerized at neutral pH and body temperature. The ability of thismaterial to gel at various concentrations was investigated. It was foundthat the digest formed a loose gel as low as 8 mg/mL. The gel was morefirm the more concentrated it was prepared. The decellularized tissuewas enzymatically digested using pepsin under acidic conditions. Thedecellularized nerve was formed into a hydrogel which spontaneouslypolymerized at neutral pH and body temperature at a concentrationbetween 8 mg/ml and 15 mg/ml (FIG. 4A-D).

Fiber length was measured by analyzing SEM images at 3000-4000 timesmagnification using Image J. Approximate fiber end-to-end lengths werefound to be 10-16 microns on average (12.1 um±3.5 um). Individual poreswere isolated and their diameters were measured using ImageJ software.Pores were randomly distributed throughout the gel and possessed averagepore diameter of 2.23±1.46 urn microns.

3.5 Restoration of Nerve Function Following Reconstruction of the RLn

Following characterization of the decellularized NS-ECM andcorresponding nerve-specific hydrogel, a pilot study evaluating theeffects of NS-ECM on anastomosis of the recurrent laryngeal nerve wasperformed. A canine preclinical model of laryngeal nerve injury, awidely accepted preclinical animal model of human laryngeal disease(Sanders (1993), Kim (2004)), was used.

The right recurrent laryngeal nerve was transected and immediatelyanastomosed, the anastomosis was surrounded by a silicone conduit andfilled with either NS-ECM or UBM hydrogel (15 mg/ml, FIG. 10A) or leftempty (control). Surgical implantation was straightforward with rapidgelling of both hydrogels within the conduit.

Six months after implantation, laryngeal endoscopy revealed arytenoidmovement but ineffective abduction, due to synkinetic reinnervationfollowing anastomosis of the common RLn trunk. Under a terminalanesthetic episode, motor action potential conduction across theanastomosis site was determined using supramaximal Rln proximalstimulation and Compound Motor Action Potential (CMAP) recording at thevocalis muscle (FIG. 10C)

Both NS-ECM and UBM significantly increased the amplitude of stimulationreaching the vocalis muscle (0.744 mV and 0.719 mV respectively)compared to the conduit alone (0.343 mV) (FIG. 10B).

Minimum Fiber diameter was increased in the NSECM and UBM groupscompared to controls in collagen V immuno-labelled right posteriorcricoarytenoid (PCA) muscles (FIG. 10D). Differences between the controland treatment groups (NS-ECM and UBM) are small but significant, 53.5 μmcompared to 58.4 μm and 61.7 μm respectively. Together these datasuggest that NS-ECM and UBM hydrogels improve nerve repair withanastomosis.

4. Discussion

4.1 Effective Decellularization

Decellularization of xenogeneic tissue offers a number of potentialbenefits: the minimization of immunogenic reactions that would lead tograft rejection, the provision of an optimized biomaterial withoutcompeting donor cells, and a reduced risk of disease transmission.However, any processing step intended to remove cells will alter thenative three-dimension architecture of the ECM to some degree. Thereforeit is generally desirable to use the mildest protocol possible thatyields an acellular material with the least disruption to the structuraland functional components of the ECM. The decellularization method usedin this Example produced an 85% decrease in DNA content when compared tonative tissue (85%; 158.1±34.5 ng/mg, 1,043.65±291.20 ng/mg).

The above-presented results demonstrate effective generation of NS-ECMhydrogel, and that NS-ECM hydrogel retains microarchitecture andpromotes dose-dependent neurite extension (bioactive). The results alsoshow that NS-ECM is a practical solution for supporting nerve repair,e.g., gels rapidly (within 60 seconds) and is readily injected intoconduit at site of injury.

4.2 Determination of Reinnervation of the Vocalis Muscle Using theAbove-Described Method

Supramaximal proximal nerve stimulation and recording of a compoundmotor action potential at the vocalis muscle allows quantification ofreinnervation across the anastomosis site. Reinnervation of thethyroarytenoid complex is crucial to the restoration of vocal cordfunction and voice. Although posterior cricoarytenoid muscle fiberdiameters were increased in both treated groups, functional recover waspoor with un-coordinated arytenoid movement and little abduction wasachieved. This result is expected with repair to the common trunk of theRLn in which abductor and adductor fibers are spatially intermingled(Crumley (2000), Benjamin (2003), Zealear (2006)).

Manipulation of the microenvironment at the site of nerve injurypromotes improved repair. An inert but non-absorbable silicone conduitwas used in this Example to maintain this microenvironment. Multipleinterventions have been shown to improve axonal regrowth in rodentmodels, however, the translation to clinical therapies in human patientshas been slow. A preclinical large animal model of laryngeal nerveinjury was used in this Example to assess NS-ECM. One of the advantagesof this approach is that nerve regrowth in large animal models is slowerand less regenerative than in rodent systems. Even the gold standard ofcare for nerve gap defects, the autologous nerve graft is associatedwith various clinical complications, including donor site morbidity,limited availability, nerve site mismatch, and the formation ofneuromas.

Scaffold based approaches using individual extracellular matrix proteinssuch as keratin, collagen and fibronectin have also been shown tosupport axonal regrowth (Hill (2011); Madison (1985); Madison (1988);Toba (2001); Lee (2006); Sierpinski (2008a)) and enhance Schwann cellfunction (Sierpinski (2008b)). Here, the complete milieu of NS-ECMproducts to promote repair was recruited.

The ECM is the framework including both structural and functionalproteins that provides the environment that cells live in. The ECMprovides cues for cell attachment, migration, and proliferation but alsois manipulated by resident cells. This dynamic interaction between ECMand cells is key for tissue development and homeostasis. Because ofthese important functions, biological scaffolds composed of ECM havebeen used in a wide variety of tissue engineering and regenerativemedicine applications, including ventral hernia repair, musculotendinoustissue reconstruction, dura mater replacement, reconstructive breastsurgery, pelvic floor reconstruction, and the treatment of cutaneousulcers, among others. These materials act as inductive templates for thegeneration of new functional, site-appropriate tissue formation (Brown(2013); Badylak (2014)).

An inductive, ECM based scaffold approach was investigated as a solutionto this problem. The advantage of using a decellularized tissue or ECMscaffold is that it promotes constructive remodeling by initiallyproviding an acellular structure that degrades rapidly, releasingmitogenic and chemotactic proteins. These modulate the innate immunesystem to promote a friendly rather than destructive immune response. Asa whole, it encourages the formation of new functional tissue (Sobotka(2011)).

ECM scaffolds derived through the decellularization of a wide array oftissues and organs have been successfully to treat injuries in varietyof preclinical and clinical applications, including skeletal muscle, theesophagus, and lower urinary tract, among others (Wang (2011a)). Recentevidence suggests that ECM scaffolds derived from site-specific tissuecan elicit a superior functional recovery in some applications (Wang(2011b)). The presence of a site specific advantage was investigated bycomparing the peripheral nerve based scaffold used in this Example tourinary bladder matrix, which is a widely used biologic scaffold.

Not all of the proposed decellularization methods have been shown to beeffective for removal of sufficient cellular content and maintenance oftissue structure, resulting in ineffective recovery in some studies. Forthis reason, histologic, immunologic, and quantitative methods was usedto validate the decellularization process.

A peripheral nerve based hydrogel was derived, characterized and testedin a canine model of laryngeal nerve transection and reconstruction inthis Example. A peripheral nerve was decellularized maintainingperipheral NS-ECM components, including nerve specific growth factors.The decellularized tissue was enzymatically digested into a mixture ofdegradation peptides. That mixture of peptides was found to be bioactiveand to promote neurite outgrowth. The peptide mixture was then formedinto a hydrogel and injected it into a clinically relevant model ofnerve transection. It was found that the NS-ECM hydrogel improvesrecovery with anastomosis.

Example 4

Animals as described in Example 3 were anesthetized and placed in leftlateral recumbency and a lateral approach to the larynx was made in astandard fashion anterior to the right linguofacial vein. Thesubcutaneous fascia was divided and the main trunk of the right RLn wasidentified. The RLn was identified and transected 15 mm inferior to thecricoid. The incisions were then closed in layers and animals wereallowed to develop chronic denervation of the RLn for three months.After three months, the animals were again anesthetized as describedabove and a nerve graft was performed. The cervical nerve was grafted tothe recurrent laryngeal nerve by anastomosis of the two ends of eachnerve. The anastomosis was surrounded by a preplaced empty 7 mm siliconeconduit (3.5 mm internal diameter, Silastic; Dow Corning, Midland,Mich.) tagged to the perineurium at either end. As shown in FIG. 11, inthe treatment group, the conduit was filled with ECM derived from equinemotor nerve (NS-ECM introduced into the conduit lumen via an 18 gaugeneedle). Hydrogels were brought to room temperature 5 minutes prior toinjection into the conduit. The incision was then closed in layers.

These data demonstrate that NS-ECM can be used to support nerve graftingeither soon after injury (acute) or after a period of delay (chronic).

7. REFERENCES

-   1. Nagao R J, Lundy S, Khaing Z Z, Schmidt C E., Neurol Res. 2011    July; 33(6):600-8-   2. Hudson T W, Zawko S, Deister C, Lundy S, Hu C Y, Lee K, Schmidt C    E, Tissue Eng. 2004 November-December; 10(11-12):1641-51.-   3. Hudson T W, Liu S Y, Schmidt C E. Tissue Eng. 2004    September-October; 10(9-10):1346-58.-   4. Medberry C J, Crapo P M, Siu B F, Carruthers C A, Wolf M T,    Nagarkar S P, Agrawal V, Jones K E, Kelly J, Johnson S A, Velankar S    S, Watkins S C, Modo M, Badylak S F., Biomaterials. 2013 January;    34(4):1033-40.-   5. Crapo P M, Medberry C J, Reing J E, Tottey S, van der Merwe Y,    Jones K E, Badylak S F., Biomaterials. 2012 May; 33(13):3539-47.-   6. Wolf M T, Daly K A, Brennan-Pierce E P, Johnson S A, Carruthers C    A, D'Amore A, Nagarkar S P, Velankar S S, Badylak S F.,    Biomaterials. 2012 October; 33(29):7028-38.-   7. U. S. Pat. No. 8,361,503 B2-   8. PCT/US2012/045682-   9. Brown B N, Badylak S F. Extracellular matrix as an inductive    scaffold for functional tissue reconstruction. Transl Res. 2013    November 8. pii: S1931-5244(13)00382-4.-   10. Badylak S F. Decellularized Allogeneic and Xenogeneic Tissue as    a Bioscaffold for Regenerative Medicine: Factors that Influence the    Host Response. Ann Biomed Eng. 2014 Jan. 9. [Epub ahead of print]-   11. Nichols, C. M., Brenner, M. J., Fox, I. K., Tung, T. H.,    Hunter, D. A., Rickman, S. R. & Mackinnon, S. E. Effects of motor    versus sensory nerve grafts on peripheral nerve regeneration. Exp.    Neurol. 190, 347-355 (2004).-   12. Lee, S. K. & Wolfe, S. W. Peripheral nerve injury and repair. J.    Am. Acad. Orthop. Surg. 8, 243-252 (2000).-   13. Mackinnon, S. E., Doolabh, V. B., Novak, C. B. & Trulock, E. P.    Clinical outcome following nerve allograft transplantation. Plast.    Reconstr. Surg. 107, 1419-1429 (2001).-   14. Kingham, P. J., Birchall, M. A., Burt, R., Jones, A. &    Terenghi, G. Reinnervation of laryngeal muscles: a study of changes    in myosin heavy chain expression. Muscle Nerve 32, 761-766 (2005).-   15. Wang, W., Chen, D., Chen, S., Li, D., Li, M., Xia, S. &    Zheng, H. Laryngeal reinnervation using ansa cervicalis for thyroid    surgery-related unilateral vocal fold paralysis: a long-term outcome    analysis of 237 cases. PLoS One 6, e19128 (2011a). PMCID:    PMC3084757.-   16. Wang, W., Chen, S., Chen, D., Xia, S., Qiu, X., Liu, Y. &    Zheng, H. Contralateral ansa cervicalis-to-recurrent laryngeal nerve    anastomosis for unilateral vocal fold paralysis: A long-term outcome    analysis of 56 cases. Laryngoscope 121, 1027-1034 (2011b).-   17. Sobotka, S. & Mu, L. Force characteristics of the rat    sternomastoid muscle reinnervated with end-to-end nerve repair. J.    Biomed. Biotechnol. 2011, 173471 (2011). PMCID: PMC3238804.-   18. Lorenz, R. R., Esclamado, R. M., Teker, A. M., Strome, M.,    Scharpf, J., Hicks, D., Milstein, C. & Lee, W. T. Ansa    cervicalis-to-recurrent laryngeal nerve anastomosis for unilateral    vocal fold paralysis: experience of a single institution. Ann. Otol.    Rhinol. Laryngol. 117, 40-45 (2008).-   19. Smith, M. E., Roy, N. & Stoddard, K. Ansa-RLN reinnervation for    unilateral vocal fold paralysis in adolescents and young adults.    Int. J. Pediatr. Otorhinolaryngol. 72, 1311-1316 (2008).-   20. Aynehchi, B. B., McCoul, E. D. & Sundaram, K. Systematic review    of laryngeal reinnervation techniques. Otolaryngol. Head. Neck.    Surg. 143, 749-759 (2010).-   21. Birchall, M. A., Lorenz, R. R., Berke, G. S., Genden, E. M.,    Haughey, B. H., Siemionow, M. & Strome, M. Laryngeal transplantation    in 2005: a review. Am. J. Transplant. 6, 20-26 (2006).-   22. Hill, P. S., Apel, P. J., Barnwell, J., Smith, T., Koman, L. A.,    Atala, A. & Van Dyke, M. Repair of peripheral nerve defects in    rabbits using keratin hydrogel scaffolds. Tissue Eng. Part A. 17,    1499-1505 (2011).-   23. Madison, R., Da Silva, C. F., Dikkes, P., Chiu, T. H. &    Sidman, R. L. Increased rate of peripheral nerve regeneration using    bioresorbable nerve guides and a laminin-containing gel. Exp.    Neural. 88, 767-772 (1985).-   24. Madison, R. D., Da Silva, C. F. & Dikkes, P. Entubulation repair    with protein additives increases the maximum nerve gap distance    successfully bridged with tubular prostheses. Brain Res. 447,    325-334 (1988).-   25. Toba, T., Nakamura, T., Shimizu, Y., Matsumoto, K., Ohnishi, K.,    Fukuda, S., Yoshitani, M., Ueda, H., Hori, Y. & Endo, K.    Regeneration of canine peroneal nerve with the use of a polyglycolic    acid-collagen tube filled with laminin-soaked collagen sponge: a    comparative study of collagen sponge and collagen fibers as filling    materials for nerve conduits. J. Biomed. Mater. Res. 58, 622-630    (2001).-   26. Lee, D. Y., Choi, B. H., Park, J. H., Zhu, S. J., Kim, B. Y.,    Huh, J. Y., Lee, S. H., Jung, J. H. & Kim, S. H. Nerve regeneration    with the use of a poly(l-lactide-co-glycolic acid)-coated collagen    tube filled with collagen gel. J. Craniomaxillofac. Surg. 34, 50-56    (2006).-   27. Sierpinski, P., Garrett, J., Ma, J., Apel, P., Klorig, D.,    Smith, T., Kaman, L. A., Atala, A. & Van Dyke, M. The use of keratin    biomaterials derived from human hair for the promotion of rapid    regeneration of peripheral nerves. Biomaterials 29, 118-128 (2008a).-   28. Sierpinski, P., Garrett, J., Ma, J., Apel, P., Klorig, D.,    Smith, T., Koman, L. A., Atala, A. & Van Dyke, M. The use of keratin    biomaterials derived from human hair for the promotion of rapid    regeneration of peripheral nerves. Biomaterials 29, 118-128 (2008b).-   29. Sanders, I., Jacobs, I., Wu, B. L. & Biller, H. F. The three    bellies of the canine posterior cricoarytenoid muscle: implications    for understanding laryngeal function. Laryngoscope 103, 171-177    (1993).-   30. Zealear, D. L., Billante, C. R., Chongkolwatana, C., Rho, Y. S.,    Hamdan, A. L. & Herzon, G. D. The effects of chronic electrical    stimulation on laryngeal muscle physiology and histochemistry.    ORL J. Otorhinolaryngol. Relat. Spec. 62, 81-86 (2000).-   31. Broome, C., Burbidge, H. M. & Pfeiffer, D. U. Prevalence of    laryngeal paresis in dogs undergoing general anaesthesia. Aust.    Vet. J. 78, 769-772 (2000).-   32. Woodson, G. E., Hughes, L. F. & Helfert, R. Quantitative    assessment of laryngeal muscle morphology after recurrent laryngeal    nerve injury: right vs. left differences. Laryngoscope 118,    1768-1770 (2008).-   33. Jansson, N., Ducharme, N. G., Hackett, R. P. & Mohammed, H. O.    An in vitro comparison of cordopexy, cordopexy and laryngoplasty,    and laryngoplasty for treatment of equine laryngeal hemiplegia. Vet.    Surg. 29, 326-334 (2000).-   34. Ducharme, N. G., Cheetham, J., Sanders, I., Hermanson, J. W.,    Hackett, R. P., Soderholm, L. V. & Mitchell, L. M. Considerations    for pacing of the cricoarytenoid dorsalis muscle by neuroprosthesis    in horses. Equine Vet. J. 42, 534-540 (2010).-   35. Goslin, K., Schreyer, D. J., Skene, J. H. & Banker, G. Changes    in the distribution of GAP-43 during the development of neuronal    polarity. J. Neurosci. 10, 588-602 (1990).-   36. Triolo, D., Dina, G., Lorenzetti, I., Malaguti, M., Morana, P.,    Del Carro, U., Comi, G., Messing, A., Quattrini, A. &    Previtali, S. C. Loss of glial fibrillary acidic protein (GFAP)    impairs Schwann cell proliferation and delays nerve regeneration    after damage. J. Cell. Sci. 119, 3981-3993 (2006).-   37. Cheetham, J., Radcliffe, C. R., Ducharme, N. G., Sanders, I.,    Mu, L. & Hermanson, J. W. Neuroanatomy of the equine dorsal    cricoarytenoid muscle: Surgical implications. Equine Vet. J. 40,    70-75 (2008).-   38. Crumley, R. L. Laryngeal synkinesis revisited. Ann. Otol.    Rhinol. Laryngol. 109, 365-371 (2000).-   39. Affleck B D, Swartz K, Brennan J 2003 Surgical consideration and    controversies in thyroid and parathyroid surgery. Otolaryngol Clin    North Am 36:159-187 Spector B C, Netterville J L, Billante C, Clary    J, Reinisch L, Smith T L 2001 Quality-of-life assessment in patients    with unilateral vocal cord paralysis. Otolaryngol Head Neck Surg    125:176-182-   40. Laccourreye O, Crevier-Buchman L, Pacona R, Ageel M, Brasnu D    1999 Intracordal fat injection for aspiration after recurrent    laryngeal nerve paralysis. Eur Arch Otoloaryngol 256:458-461-   41. Negus V E 1929 The mechanism of the larynx. London: W. M.    Heinemann Ltd.; 1-45-   42. Wolf, M., Daly, K., Reing, J., & Badylak, S. (2012). Biologic    scaffold composed of skeletal muscle extracellular matrix.    Biomaterials, 33(10), 2916-25.-   43. Sawkings, M., Bowen, W., Dhadda, P., Markides, H., Sidney, L.,    Taylor, A., & White, L. (2013). Hydrogels derived from demineralized    and decellularized bone extracellular matrix. 9(8), 7865-73.-   44. Badylak, S. (2011, October). Regenerative medicine:    Possibilities and potential. The singularity summit.-   45. Dralle H. Intraoperative monitoring of the recurrent laryngeal    nerve in thyroid surgery. World J Surg. 2008 32(7): 1358-1366.-   46. Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and    whole organ decellularization processes. Biomaterials. 2011 April;    32(12):3233-43.-   47. Gilbert T W, Freund J M, Badylak S F. Quantification of DNA in    biologic scaffold materials. J Surg Res. 2009 March; 152(1):135-9.-   48. Brown B, Lindberg K, Reing J, Stolz D B, Badylak S F. The    basement membrane component of biologic scaffolds derived from    extracellular matrix. Tissue Eng. 2006 March; 12(3):519-26.-   49. Kim, M. J., Hunter, E. J. & Titze, I. R. Comparison of human,    canine, and ovine laryngeal dimensions. Ann. Otol. Rhinol. Laryngol.    113, 60-68 (2004).-   50. Benjamin, B. Vocal cord paralysis, synkinesis and vocal fold    motion impairment. ANZ J Surg 73, 784-786 (2003).-   51. Zealear, D. L. & Billante, C. R. In: Vocal Fold Paralysis (eds    Sulica, L. & Blitzer, A.) 17-32 (Springer Berlin, Heidelberg, 2006).-   52. Deal D N, Griffin J W, Hogan M V, Nerve conduits for nerve    repair or reconstruction. J Am Acad Orthop Surg. 2012 February;    20(2):63-8.

Various references are cited herein, the contents of which are herebyincorporated by reference in their entireties.

We claim:
 1. A method of repairing a nerve injury, comprising injectinga hydrogel comprising a decellularized peripheral nerve scaffold thathas been enzymatically degraded, so as to bridge a gap in the nerve. 2.The method of claim 1, where the gap is a critically sized defect. 3.The method of claim 1, where the hydrogel is injected into a conduit. 4.The method of claim 2, where the hydrogel is injected into a conduit. 5.The method of claim 1, where the peripheral nerve is non-autologous toan intended recipient of the hydrogel.
 6. The method of claim 5, wherethe peripheral nerve is from an organism that is the same species as theintended recipient.
 7. The method of claim 5, where the peripheral nerveis from an organism that is not the same species as the intendedrecipient.
 8. A method of promoting incorporation of a graft to repair agap in a nerve, comprising providing, into the gap, a graft and ahydrogel comprising a decellularized peripheral nerve scaffold that hasbeen enzymatically degraded.
 9. The method of claim 8, where the gap isa critically sized defect.
 10. The method of claim 8, where the hydrogelis injected into a conduit.
 11. The method of claim 9, where thehydrogel is injected into a conduit.
 12. The method of claim 8, wherethe peripheral nerve is non-autologous to an intended recipient of thehydrogel.
 13. The method of claim 12, where the peripheral nerve is froman organism that is the same species as the intended recipient.
 14. Themethod of claim 12, where the peripheral nerve is from an organism thatis not the same species as the intended recipient.